How the huge neuronal diversity observed in our brain is generated and how these neurons are assembled into functional networks are poorly understood. A unique model to study these complex questions is the Drosophila mushroom body (MB), which processes olfactory learning and memory, because its basic function and development are well characterized. Each MB consists of five cell types that are born sequentially from four identical neuroblasts: first, 'non-intrinsic' (ni) neurons that do not participate to the MB are born, then ?, ?'?', pioneer-?? and finally ?? neurons are generate. Vertebrate neural progenitors also produce neuron types sequentially, indicating that temporal patterning is conserved. Drosophila NBs have emerged as an excellent model to study the molecular mechanisms that regulate temporal patterning. We hypothesize that a temporal transcription factor cascade functions in MB neuroblasts to define each MB neuronal type during development. We also hypothesize that, unlike in most brain structures, MB intermediate precursors divide symmetrically to produce two identical neurons to generate the large number of identical neurons necessary to perform MB functions. We will determine the molecular mechanisms regulating the temporal and symmetrical production of MB cell types. This will provide novel insights into developmental programs generating the diverse neurons that make up our brains. Aim 1: Define and test the mechanisms that sequentially specify MB neuron types. The sequential production of neurons from MB NBs involves four transitions resulting in five distinct cell types. We hypothesize that MB neuroblasts sequentially express a series of transcription factors as they age to confer neuronal identity. We will identify these transcription factors and use mutant clonal analysis, RNAi and overexpression assays to address the function of these factors in producing MB neuron types. Aim 2: Study the regulation of asymmetric NB and symmetric GMC division in the MB. Although MB neuroblasts divide asymmetrically, MB intermediate precursors divide symmetrically to generate two identical neurons. In all other studied lineages, these cells divide asymmetrically to generate a NotchON and a NotchOFF neuron. We will test whether both identical MB neurons are NotchOFF or NotchON as a consequence of rotating the axis of the last cell division orthogonal to the MB neuroblast division plane. Aim 3: Determine the RNA profile of mature MB neuron types. We have Gal4 lines that mark individual MB neuron types. We will use them and others we identify to FACS and then transcriptionally profile adult ?, ?'?', pioneer-?? and ?? neurons to understand the specification of these neurons. Adult neurons may also maintain expression of the transcription factors that specify the temporal windows of MB neuroblasts. Expression and function of candidate genes differentially expressed between each MB neuron type will be tested.